Although progress has been made in understanding the role of follicle stimulating hormone (FSH) in governing the process of preovulatory folliculogenesis, a major question that remains unanswered is the elucidation of the intracellular signaling pathways utilized by FSH in the regulation of the complex pattern of gene expression that occurs during follicular development. Our preliminary results point to two loci through which FSH may optimally induce granulosa cell differentiation. First, activation of a pathway or pathways in addition to PKA synergizes with the PKA pathway to optimally induce the expression of a subset of genes including aromatase and the LH receptor, the two hallmark genes associated with granulosa cell differentiation. Second, compartmentalization of the FSH/cAMP signaling pathway may result in more efficient coupling of intracellular cAMP production with the induction of the estrogen and progesterone biosynthetic pathways. In this current proposal we present four Specific Aims to address these findings. Aim 1 will compare the signaling pathways activated by FSH and by PKA-CQR in undifferentiated granulosa cells. Aim 2 will determine which signaling pathways and/or transcription factors interact with PKA to optimally induce granulosa cell differentiation. Aim 3 will explore the possible intracellular compartmentalization of the cAMP/PKA signaling system in granulosa cells by disrupting PKA/AKAP interactions with HT31 peptides on granulosa cell differentiation and using novel FRET-based PKA sensors to directly evaluate compartmentalization of cAMP/PKA signaling in granulosa cells. Understanding the signaling pathways involved in granulosa cell differentiation may be instrumental for the development of novel contraceptives and fertility drugs as well as providing new information regarding signaling pathways that may be affected in anovulatory disorders such as ovarian failure and polycystic ovarian syndrome. PUBLIC HEALTH RELEVANCE: This project seeks to identify the cellular mechanisms that control the maturation of the ovarian follicle.